Assays using calibration against an internal standard

If the recovery in an assay is good and the instrumentation used for measurement of the sample is capable of high precision, the use of an internal standard is not necessary. HPLC instrumentation is usually capable of high precision but for certain samples, recoveries prior to injection into the HPLC may not be accurate or precise. Examples of formulations in which recoveries may not be complete include ointments and creams, which require more extensive extraction prior to analysis. Problems of recovery are also typical of advanced drug delivery systems, which may be based on polymeric matrices in which a drug is dispersed. An internal standard is a compound related to the analyte (the properties required for an internal standard are summarised later), which is ideally added to the formulation being analysed prior to extraction. Quantification is achieved by establishing a response factor for the analyte relative to the internal standard, i.e. a ratio for the areas of the chromatographic peaks obtained for equal amounts of the analyte and internal standard; ideally this should be close to l for equal amounts of analyte and internal standard. The response factor may be based on a single-point calibration or a full calibration curve may be constructed; all the BP assays of this type are based on single-point calibrations. Once a response factor has been established the sample is extracted with a solution containing the same concentration of internal standard as was used in determining the response factor (or a solution which after dilution will yield an extract in which the internal standard is at the same concentration as in the calibration solution). Provided the solution containing the fixed concentration of internal standard is added to the sample in a precisely measured volume, any subsequent losses of sample are compensated for since losses of the analyte will be mirrored by losses of the internal standard. The example given in Box 12.3 is typical of a BP assay incorporating an internal standard.

Box 12.2 Properties of an internal standard

• Ideally should be closely related in structure to the analyte

• Should be stable

• Should be chromatographically resolved from the analyte and any excipients present in the chromatogram of the formulation extract

• Should elute as close as possible to the analyte with the restrictions above

• For a given weight should produce a detector response similar to that produced by the analyte

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Cure Tennis Elbow Without Surgery

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